Gold-coated nanoparticles for use in biotechnology applications

ABSTRACT

A process of preparing gold-coated magnetic nanoparticles is disclosed and includes forming a suspension of magnetic nanoparticles within a suitable liquid, adding an amount of a reducible gold compound and a reducing agent to the suspension, and, maintaining the suspension for time sufficient to form gold-coated magnetic nanoparticles.

STATEMENT REGARDING FEDERAL RIGHTS

This invention was made with government support under Contract No.W-7405-ENG-36 awarded by the U.S. Department of Energy. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to gold-coated nanoparticles andparticularly gold-coated magnetic nanoparticles for use in biotechnologyapplications.

BACKGROUND OF THE INVENTION

In recent years, there has been an interest in using nanoparticles andmagnetic nanoparticles for medicinal and biomolecular applications. U.S.Pat. No. 6,470,220 by Kraus, Jr. et al. described diagnosis andtreatment of cancers using in vivo magnetic domains. This treatmentapproach employs a magnetic particle having a cancer-binding agentattached thereon. The cancer-binding agent should have bindingspecificity for the desired cancer cells. Such magnetic particles can beused as imaging agents in conjunction with magnetic resonance imaging(MRI), as a directing agent for therapeutics guided by external magneticfields and for magnetotherapy techniques.

One prime candidate for the magnetic material is samarium cobalt.However, samarium cobalt has not previously been used as an agent withinthe body and has not previously been tested for biocompatibility. Thus,necessary governmental approvals would be required prior to the use ofsamarium cobalt.

Since samarium cobalt has such desirable magnetic properties forselected therapeutics and magnetotherapy techniques, a biocompatiblyacceptable composition including a magnetic material such as samariumcobalt has been sought. After extensive and careful investigation, acomposition has now been developed, in particular, a gold-coatedmagnetic particle composition.

It is an object of the present invention to provide a process ofpreparing a gold-coated nanoparticle composition, in particular agold-coated magnetic nanoparticle composition for use in biotechnologyapplications.

It is another object of the present invention to provide a gold-coatednanoparticle composition, in particular a gold-coated magneticnanoparticle composition for use in biotechnology applications.

SUMMARY OF THE INVENTION

To achieve the foregoing and other objects, and in accordance with thepurposes of the present invention, as embodied and broadly describedherein, the present invention provides a process of preparinggold-coated magnetic nanoparticles by forming a suspension of magneticnanoparticles within a suitable liquid, adding an amount of a reduciblegold compound and a reducing agent to the suspension, maintaining thesuspension for time sufficient to form gold-coated magneticnanoparticles. In another embodiment, the process further includesreacting the gold-coated magnetic nanoparticles with amercapto-terminated bifunctional compound to form compositenanoparticles of a thiol-bound functional group-containing spacer groupthereon the gold-coated magnetic nanoparticles. In still anotherembodiment, the process further includes reacting the functional groupupon the composite nanoparticles with a linker group having oneterminally protected functionality.

The present invention further provides a gold-coated magneticnanoparticle composite including a magnetic nanoparticle central core,and, a coating of gold completely encapsulating said magneticnanoparticle central core. In another embodiment, the composite furtherincludes thiol-bound functional group-containing spacer groups thereonthe gold-coated magnetic nanoparticles. In still another embodiment, thecomposite further includes linker groups bound at one end with saidthiol-bound functional group-containing spacer groups thereon thegold-coated magnetic nanoparticles, the linker groups also having oneterminally protected functionality.

DETAILED DESCRIPTION

The present invention is concerned with preparation of coated magneticnanoparticles and a gold-coated magnetic nanoparticle composite. Suchgold-coated magnetic nanoparticle composites have significant potentialuse in biotechnology applications. A gold coating would prevent directbio-contact to the magnetic material thus improving biocompatibility.Also, a gold surface allows good coupling through chemical attachment ofdesired cancer binding agents.

By “nanoparticles” is meant, particles having dimensions of from about10 nanometers (nm) to 250 nm, more preferably from about 10 nm to about100 nm.

By “linker group” is meant to include a bifunctional molecule capable ofcovalently linking two other molecules to one another, e.g.,bifunctional organic compounds such as H₂N(CH₂)_(n)COOH orH₂N(CH₂)_(n)NH₂ where n is an integer from 1 to 12, and the like, aterminally protected bifunctional organic compound such asFmocHN(CH₂)₃NH₂ and the like, glycols such as polyalkylene glycols,e.g., polyethylene glycol (PEG) or polypropylene glycol (PPG), andcysteamines and homologues thereof.

By “mercapto-terminated bifunctional compound” is meant to includecompounds having a mercapto or thio group, typically at one end of amolecule, and another functional group such as a carboxylic acid, anamine, a sulfhydryl, a phosphate, a phosphonate hydroxyl, an alkenyl oran alkyne group, typically at another end of the molecule. Amongsuitable mercapto-terminated bifunctional compounds is3-mercaptopropionic acid.

By “recognition group” is meant to include biological recognitionelements including peptides (e.g., antibodies, antibody fragments andreceptors), oligonucleotides, nucleotides, nucleic acids, polypeptides,proteins, and oligosaccharides that specifically recognize and bind atarget molecule. In some instances, such recognition groups may bereferred to as “biologically active molecules” such molecules from thegroup of a hapten, a biologically active ligand, a drug, a peptide, anoligonucleotide, a nucleotide, a nucleic acid, a polypeptide, a protein,an antibody, an antibody fragment and the like.

Among magnetic materials for use in the present invention are includedmagnetic materials from among the elements cobalt, iron, nickel,samarium, neodymium, platinum, boron, compounds thereof and alloysthereof. Ferromagnetic materials and rare earth containing materialssuch as, e.g., iron-cobalt (Fe—Co), iron-platinum (Fe—Pt),iron-cobalt-nickel (Fe—Co—Ni), samarium-cobalt (Sm—Co),neodynium-iron-boride (Nd—Fe—B) are suitable examples. Other magneticmaterials, e.g., superparamagnetic materials such as iron oxides (Fe₃O₄)may be used as well.

In the process of the present invention, the starting materials includea reducible gold compound and a reducing agent. Among suitable reduciblegold compounds are included sodium terachloroaurate, sodiumtetrabromoaurate, tetrachloroauric acid, tetrabromoauric acid, potassiumtetrachloroaurate, and potassium tetrabromoaurate. Among suitablereducing agents are included sodium citrate, sodium borohydride, whitephosphorus, lithium aluminum hydride, and sodium cyanoborohydride.Following coating of the magnetic material with gold, the composite canbe reacted with mercapto-terminated bifunctional compounds to yieldorganic moieties upon the surface which can be modified using steps wellknown to those skilled in the art to provide suitable linkages torecognition groups and the like.

In a preferred embodiment of the present invention, samarium cobaltnanoparticles are coated with gold by admixture with a gold compoundsuch as sodium tetrachloroaurate in combination with a suitable reducingagent.

The present invention is more particularly described in the followingexamples, which are intended as illustrative only, since numerousmodifications and variations will be apparent to those skilled in theart.

EXAMPLE 1

Samarium cobalt (SmCO₅) nanoparticles (0.100 g; 0.225 mmol) weresuspended in 25 mL of water with use of a sonic dismembrator (FisherModel 60, 80–90 watts) at 0° C. Excess sodium terachloroaurate (NaAuCl₄)(0.820 g, 2.25 mmol) was added to the suspension and reduced to themetal using a freshly prepared solution of sodium borohydride (0.130 g,3.44 mmol) in 50 mL of water. The resultant gold-coated SmCO₅ particleswere separated from the co-produced gold nanoparticles by a permanentmagnet and the gold-coated SmCO₅ particles were washed with three 25-mLportions of water. The gold-coated SmCO₅ particles were dried overnightunder a high vacuum. Visual examination of the gold-coated magneticnanoparticles showed that the magnetic nanoparticles were completelycoated with a layer of gold.

EXAMPLE 2

A 15 mL centrifuge tube was charged with gold-coated SmCO₅ particles(0.1023 g) from example 1 followed by N,N-dimethylformamide (DMF) (5 mL)and 3-mercaptopropionic acid (0.7 mL, 8.0 mmol). The mixture was placedon a rotator overnight and then spun down in a centrifuge. The liquidwas decanted off and the solid was washed with three 10-mL portions ofDMF. The resultant coated-material included a free carboxylic acidgroup.

EXAMPLE 3

A 15 mL centrifuge tube with a cap was charged with the gold-coatedSmCO₅ particles having free carboxylic acid groups thereon from example2, 1-hydroxybenzotriazole(HOBt)/2-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) (0.5 M in DMF, 3 mL) anddiisopropylethylamine (2.0 M in N-methylpyrrolidone, 1.5 mL). Themixture was placed on a rotator for 30 minutes and then spun down in acentrifuge. The liquid was decanted off and the solid was washed withthree 10-mL portions of DMF. N,N-dimethylformamide (5 ml) was added tothe solid followed by addition of Fmoc-ethylenediamine hydrochloride(2.27 g, 8.04 mmol) diisopropylethylamine (2.0 M in N-methylpyrrolidone,5 mL). The mixture was placed on a rotator overnight and then spun downin a centrifuge. The liquid was decanted off and the solid was washedwith three 5-mL portions of DMF. The particles were dried overnightunder a high vacuum.

EXAMPLE 4

The Fmoc label was cleaved using piperidine and monitored by UV to givethe loading factor, i.e., the amount of accessible Fmoc groups per gramof material. The loading factor using this procedure was determined tobe 0.06 mmol Fmoc per gram of labeled SmCO₅ particles. As a comparison,loading factors for commercial solid phase peptide synthesis supportmaterial are in the range of 0.2 to 5 mmol Fmoc per gram of supportmaterial. With SmCO₅ particles having more than a five-fold higherdensity than commercially available polystyrene resin core material, theloading of the gold-coated magnetic particles was within an expectedrange.

Although the present invention has been described with reference tospecific details, it is not intended that such details should beregarded as limitations upon the scope of the invention, except as andto the extent that they are included in the accompanying claims.

1. A process of preparing gold-coated magnetic nanoparticles comprising:dispersing magnetic nanoparticles wherein said magnetic nanoparticlesare of a magnetic material selected from the group consisting of theelements samarium, neodymium, compounds thereof and alloys thereofwithin a suitable liquid to form a suspension; adding an amount of areducible gold compound and a reducing agent to the suspension; and,maintaining the suspension for time sufficient to form gold-coatedmagnetic nanoparticles wherein said gold coating is directly on saidmagnetic nanoparticles.
 2. The process of claim 1 wherein said reduciblegold compound is selected from the group consisting of sodiumtetrachloroaurate, sodium tetrabromoaurate, tetrachloroauric acid,tetrabromoauric acid, potassium tetrachloroaurate, and potassiumtetrabromoaurate.
 3. The process of claim 1 wherein said reducing agentis selected from the group consisting of sodium citrate, sodiumborohydride, white phosphorus, lithium aluminum hydride, and sodiumcyanoborohydride.
 4. The process of claim 1 further including reactingsaid gold-coated magnetic nanoparticles with a mercapto-terminatedbifunctional compound to form composite nanoparticles of a thiol-boundfunctional group-containing spacer group thereon said gold-coatedmagnetic nanoparticles.
 5. The process of claim 4 further includingreacting said functional group upon said composite nanoparticles with alinker group having one terminally protected functionality.
 6. Theprocess of claim 5 wherein said linker group having one terminallyprotected functionality is selected from the group consisting ofFmoc-ethylenediamine, ethylene glycols, propylene glycols, cysteaminesand homologues thereof.
 7. The process of claim 5 wherein said linkergroup having one terminally protected functionality isFmoc-ethylenediamine.
 8. The process of claim 4 wherein saidmercapto-terminated bifunctional compound includes as a functionalityselected from the group consisting of carboxylic acid, amine,sulfhydryl, phosphate, phosphonate hydroxyl, alkenyl, and alkyne.
 9. Theprocess of claim 4 further including de-protecting the one terminallyprotected functionality and reacting said functionality with arecognition group, a bioconjugative reactive moiety or a biologicallyactive moiety.
 10. A process of preparing gold-coated magneticnanoparticles comprising: dispersing magnetic nanoparticles of samariumcobalt within a suitable liguid to form a suspension; adding an amountof a reducible gold compound and a reducing agent to the suspension;and, maintaining the suspension for time sufficient to form gold-coatedmagnetic nanoparticles wherein said gold coating is directly on saidmagnetic nanoparticles.
 11. The process of claim 10 wherein saidreducible gold compound is selected from the group consisting of sodiumtetrachloroaurate, sodium tetrabromoaurate, tetrachloroauric acid,tetrabromoauric acid, potassium tetrachloroau rate, and potassiumtetrabromoau rate.
 12. The process of claim 10 wherein said reducingagent is selected from the group consisting of sodium citrate, sodiumborohydride, white phosphorus, lithium aluminum hydride, and sodiumcyanoborohydride.
 13. The process of claim 10 further including reactingsaid gold-coated magnetic nanoparticles with a mercapto-terminatedbifunctional compound to form composite nanoparticles of a thiol-boundfunctional group-containing spacer group thereon said gold-coatedmagnetic nanoparticles.